Methylobacterium extorquens is a pink-pigmented facultative methylotroph (PPFM) capable of growth on simple and inexpensive single-carbon compounds, such as methanol, as the sole carbon and energy source in a completely synthetic mineral salt medium (Bourque et al., 1992). These simple requirements combined with the fully automated nutrient non-limiting high cell density fed-batch bioprocesses developed for M. extorquens ATCC 55366 (Bourque et al, 1995; Bélanger et al., 2004; Beland et al., 2004), render large-scale M. extorquens fermentations very cost-effective. This feature, along with the availability of genetic tools (Marx and Lidstrom, 2001; Figueira et al., 2003; Choi et al., 2004) and abundant genome sequence information and stoichiometric models for evaluating its metabolic capabilities (Van Dien and Lidstrom, 2002), makes M. extorquens very interesting economically as a host for the production of recombinant proteins. Overexpression in M. extorquens of recombinant green fluorescent protein, esterase from Lactobacillus casei, catechol 2,3-dioxygenase from Pseudomonas putida, enterocin P from Enterococcus faecium, and haloalkane dehalogenase from Xanthobacter autotrophicus have been described in the literature (Fitzgerald and Lidstrom, 2003; Bélanger et al., 2004; Choi et al., 2004; Gutierrez et al., 2005).
Methylobacterium strains are ubiquitous in nature, inhabiting soils (Sy et al., 2001), sediments and fresh water environments (Rickard et al., 2002). Methylobacterium strains have also been detected and isolated from the surface of leaves from almost all plants tested (Romanovskaya 2001; Omer et al., 2004; Koopman and Kutschera, 2005; Gallego et al., 2005). Furthermore, there are a growing number of reports describing favourable interactions between PPFMs and plants. M. extorquens has been described as an endophytic microorganism, found in the stem and leaves of citrus plants (Lacava et al., 2004), as a bud endophyte of Scots pine (Pirttila, 2000), and in the rhizosphere of flowering plants (Idris et al., 2004).
Recently, our laboratory has successfully cloned and expressed in M. extorquens the cry1Aa gene. The toxin protein encoded by cry1Aa is highly active against the spruce budworm, a powerful forest defoliating pest. These observations in view of the ubiquitous nature of M. extorquens in the environment and the ease by which the strain can be genetically transformed to over-express recombinant proteins, suggests that this microorganism could be utilized as an attractive delivery system of recombinant biocontrol agents against crop and forest insect pests. However, the utilization of expression systems in the environment or under high cell density fermentation bioprocesses can be problematic. The reason for this being plasmid segrational instability in the absence of selective pressure. The loss of plasmid and consequently the loss of expression of the desired recombinant product occurs gradually but surely under growth conditions where antibiotics cannot be used for practical or safety issues. We have shown in a previous study (Bélanger et al., 2004) that approximately 50% of recombinant expression is lost following 15 generations of growth in the absence of selective pressure in M. extorquens. Integration of expression cassettes in bacterial chromosomes would not necessitate the use of antibiotic selection for the stable expression of recombinant products. It has been recently shown that the Tn7 system integrates, in a stable and site-specific manner, recombinant DNA fragments into a site of the chromosome called attTn7. This attTn7 is located in the intergenic region downstream of the glmS gene in many Gram negative bacteria such as E. coli, Klebsiella pneumonia, Serratia marcescens, Pseudomonas putida and Yersinia pestis (Craig, 1989; Lichtenstein and Brenner, 1982; Choi et al., 2005).